RESUMO
Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron-chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non-ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC-MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl-coenzyme A (CoA)-hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)-CoA synthase Hcs1 in U. maydis that HMG-CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.
Assuntos
Vias Biossintéticas/genética , Ferricromo/análogos & derivados , Ustilago/genética , Ustilago/metabolismo , Acil Coenzima A/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , Ferricromo/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Expressão Gênica , Ordem dos Genes , Genes Fúngicos , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Espectrometria de Massas , Modelos Biológicos , Estrutura Molecular , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The opportunistic human pathogenic fungus Aspergillus fumigatus is a major cause of fungal infections in immunocompromised patients. Innate immunity plays an important role in the defense against infections. The complement system represents an essential part of the innate immune system. This cascade system is activated on the surface of A. fumigatus conidia and hyphae and enhances phagocytosis of conidia. A. fumigatus conidia but not hyphae bind to their surface host complement regulators factor H, FHL-1, and CFHR1, which control complement activation. Here, we show that A. fumigatus hyphae possess an additional endogenous activity to control complement activation. A. fumigatus culture supernatant efficiently cleaved complement components C3, C4, C5, and C1q as well as immunoglobulin G. Secretome analysis and protease inhibitor studies identified the secreted alkaline protease Alp1, which is present in large amounts in the culture supernatant, as the central molecule responsible for this cleavage. An alp1 deletion strain was generated, and the culture supernatant possessed minimal complement-degrading activity. Moreover, protein extract derived from an Escherichia coli strain overproducing Alp1 cleaved C3b, C4b, and C5. Thus, the protease Alp1 is responsible for the observed cleavage and degrades a broad range of different substrates. In summary, we identified a novel mechanism in A. fumigatus that contributes to evasion from the host complement attack.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/imunologia , Complemento C3/metabolismo , Complemento C4/metabolismo , Complemento C5/metabolismo , Proteínas Fúngicas/metabolismo , Serina Endopeptidases/metabolismo , Animais , Aspergillus fumigatus/genética , Complemento C1q/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Deleção de Genes , Humanos , Hifas/enzimologia , Hifas/imunologia , Evasão da Resposta Imune , Imunoglobulina G/metabolismo , Camundongos , Serina Endopeptidases/genéticaRESUMO
The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. We established a 2-D reference map for A. fumigatus. Using MALDI-TOF-MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.
Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/análise , Proteoma/análise , Aspergillus fumigatus/citologia , Ciclo Celular , Proteínas Fúngicas/metabolismo , Humanos , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
With the completion of the Aspergillus fumigatus genome it is now possible to study protein regulation on a global scale. One of the most suitable protein separation techniques is based on 2D-gel electrophoresis, which allows the separation of proteins based on their charge and size in a gel matrix. In addition, gel-free proteomics techniques based on liquid-chromatography coupled with mass spectrometry have gained importance. With the application of proteomic tools a comprehensive overview about the proteins of A. fumigatus present or induced during environmental changes and stress conditions can be obtained. For A. fumigatus, several proteomic studies have already been published including the response of the fungus to oxidative stress that induced the up-regulation of many proteins including catalases and thioredoxin peroxidase. Since many of the identified proteins/genes were apparently regulated by a putative Saccharomyces cerevisiae Yap1 homolog, the corresponding gene of A. fumigatus was identified, designated Afyap1 and further characterized. In addition, some of the gene products expressed under stress conditions are also known fungal antigens, such as the thioredoxin peroxidase AspF3. Thus, besides pathogenicity studies, proteomics also delivers the tools to screen for new antigens which could improve the diagnosis of diseases caused by A. fumigatus.
Assuntos
Aspergillus fumigatus/química , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/análise , Proteoma/análise , Fatores de Virulência/análise , Aspergillus fumigatus/fisiologia , Regulação Fúngica da Expressão Gênica , Estresse FisiológicoRESUMO
Macrophages and neutrophils kill the airborne fungal pathogen Aspergillus fumigatus. The dependency of this killing process on reactive oxygen intermediates (ROI) has been strongly suggested. Therefore, we investigated the enzymatic ROI detoxifying system by proteome analysis of A. fumigatus challenged by H(2)O(2). Since many of the identified proteins and genes are apparently regulated by a putative Saccharomyces cerevisiae Yap1 homolog, the corresponding gene of A. fumigatus was identified and designated Afyap1. Nuclear localization of a functional AfYap1-eGFP fusion was stress dependent. Deletion of the Afyap1 gene led to drastically increased sensitivity of the deletion mutant against H(2)O(2) and menadione, but not against diamide and NO radicals. Proteome analysis of the DeltaAfyap1 mutant strain challenged with 2 mM H(2)O(2) indicated that 29 proteins are controlled directly or indirectly by AfYap1, including catalase 2. Despite its importance for defense against reactive agents, the Afyap1 deletion mutant did not show attenuated virulence in a murine model of Aspergillus infection. These data challenge the hypothesis that ROI such as superoxide anions and peroxides play a direct role in killing of A. fumigatus in an immunocompromised host. This conclusion was further supported by the finding that killing of A. fumigatus wild-type and DeltaAfyap1 mutant germlings by human neutrophilic granulocytes worked equally well irrespective of whether the ROI scavenger glutathione or an NADPH-oxidase inhibitor was added to the cells.
Assuntos
Aspergillus fumigatus/metabolismo , Regulação Fúngica da Expressão Gênica , Granulócitos/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Aspergillus fumigatus/patogenicidade , Feminino , Granulócitos/microbiologia , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neutrófilos/microbiologia , Homologia de Sequência de AminoácidosRESUMO
In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem.
Assuntos
Ferro/metabolismo , Ustilago/patogenicidade , Zea mays/microbiologia , Transporte Biológico , Membrana Celular/metabolismo , Mapeamento Cromossômico , AMP Cíclico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Oxirredução , Proteínas Recombinantes de Fusão/metabolismo , Ustilago/citologia , VirulênciaRESUMO
Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. One of the important questions concerning A. fumigatus is the identification of pathogenicity determinants. To obtain a comprehensive overview about the proteins produced at different physiological conditions that are related to the infectious process a proteomic approach has been applied. Here, 2-D gel electrophoresis for filamentous fungi was optimised concerning removal of interfering compounds, protein extraction and separation methods. A trichloroacetic acid-based precipitation method of proteins with their subsequent solubilisation by the use of a combination of CHAPS with a second sulfobetaine detergent gave the best results. The optimised protocol was evaluated by the analysis of the proteomes of A. fumigatus grown on two different carbon sources, i.e., glucose and ethanol. Carbon catabolite repression has not been studied in detail at the protein level in A. fumigatus yet. In addition, growth on ethanol leads to activation of the glyoxylate cycle which was shown to be essential for pathogenesis in bacteria and fungi. In A. fumigatus, differential patterns of enzymes of the gluconeogenesis, glyoxylate cycle and ethanol degradation pathway during growth on glucose and ethanol were observed.